紫色大花矮牵牛组织培养与植株再生

矮牵牛叶片外植体在MS+6-BA 1.0mg/L+NAA 0.1mg/L培养基上培养3周后产生致密的浅绿色愈伤组织;转入芽分化培养基MS+6-BA 0.5mg/L+4-PU 0.5mg/L+NAA 0.1mg/L 1周后,从愈伤组织表面不断分化产生幼芽;待幼芽长至3cm时转接至生根培养基1/2MS+NAA 1.0 mg/L+GA₃0.5mg/L中生根,长成完整植株。     

罗汉果叶片离体再生快繁技术

以罗汉果(Ssiraiti grosvenori)叶片为外植体,探讨培养方式、激素组合对愈伤组织诱导和不定芽分化的影响。结果表明:罗汉果叶片在培养基MS+BA 1.0 mg/L+IBA 0.7 mg/L上培养4周愈伤组织的诱导率达90%以上;罗汉果愈伤组织在培养基MS+BA 1.0 mg/L+IBA0.2 mg/L+赛苯隆(TDZ)0.1 mg/L上不定芽的分化率可达70%,平均出芽指数3.7;罗汉果试管苗在培养基MS+BA 2.0 mg/L+IBA 0.2 mg/L上茎芽增殖比较稳定,在培养基MS+IBA0.1 mg/L上培养2周开始分化不定根,其生根率在90%以上。     

药用植物栝楼的组织培养及其表达蛋白的分析

对栝楼的快速繁殖、愈伤组织的诱导与再分化,以及不同培养体系中天花粉蛋白的表达进行了初步研究。结果表明：栝楼茎切段的腋芽和顶芽在MS+0.5、1.0mg/L 6-BA培养基上可以快速繁殖;组织培养苗的叶片切块在MS+4.0mg/L 6-BA +0.2mg/L IAA的培养基上可形成愈伤组织,该愈伤组织在30d后再分化为绿苗,绿苗分化率为0.25苗/外植体;绿苗转移至MS+0.1mg/L NAA的培养基可100%生根;生根苗移栽至土壤中100%成活;移栽成活的栝楼在30d后长出小块根,并检测到天花粉蛋白的表达。     

红叶石楠离体植株再生频率的影响因素研究

以红叶石楠带芽茎段及叶片为外植体,分析激素和培养条件等因子对愈伤组织诱导及植株再生的影响。结果表明,MS+0.10mg/L 2,4-D+0.50mg/L NAA+0.50mg/L 6-BA+0.50mg/L KT为最佳愈伤组织诱导培养基,暗培养的愈伤组织诱导率高于光培养,其愈伤组织诱导率可达100%(带芽茎段)和98%(叶片)。MS+0.50mg/L IBA+2.00mg/L 6-BA+2.00mg/L KT为最佳分化增殖培养基,分化率91%以上,增殖倍数6.8以上,均达到最高。1/2MS+0.50mg/L IBA+0.01mg/L NAA为最佳生根培养基,生根率92%,生根量4.4根/株,均达到最高。     

如意皇后粗肋草的离体培养

本研究以粗肋草‘Red Valentine’带侧芽的根茎为外植体,研究不同消毒时间、不同种类外源激素对其根茎侧芽萌发诱导、愈伤组织诱导、丛生芽诱导、丛生芽增殖和生根的影响。结果表明:最佳灭菌时间为18 min,污染率为20.0%。MS+6-BA 2.0 mg/L+IBA 0.2 mg/L为最佳侧芽萌发诱导培养基,MS+TDZ 0.5 mg/L+2,4-D 2.0 mg/L为最优愈伤诱导培养基,1/2MS+TDZ 0.5 mg/L为最佳丛生芽诱导培养基,最佳丛生芽增殖培养基为MS+6-BA 3.0 mg/L+NAA 0.1 mg/L或MS+KT 4.0 mg/L+NAA 0.1 mg/L,最佳生根培养基为1/2MS+NAA 0.2 mg/L。本研究通过对粗肋草‘Red Valentine’进行离体培养,初步得出了适合外植体各时期生长的最佳培养基配方,为粗肋草试管苗的商业化、工厂化及规模化生产提供理论和技术指导。     

青蒿组织培养及其快速繁殖研究

以青蒿幼叶、叶柄为外植体,研究其离体培养和试管苗再生途径。结果表明,以青蒿嫩叶为外植体,在MS+6-BA0.5mg/L+IBA0.5mg/L的培养基中可诱导出愈伤组织,诱导率达87%,并在此培养基中可以分化出芽,分化率为85%,将分化苗转移到MS+IBA0.5mg/L的培养基上,生根率高达93%。     

玉竹的组织培养与快速繁殖

以玉竹[Polygonatum odoratum (Mill.) Druce]根状茎、叶片和茎段为外植体,于附加不同激素配比的MS培养基中诱导愈伤组织、不定芽和不定根,探讨增殖培养和植株再生的条件.结果表明,叶片和茎段外植体诱导愈伤组织和芽的分化率很低;而根状茎外植体易于培养,有较高的诱导率和增殖倍数,其愈伤组织、不定芽和不定根的诱导率分别可达87%、90%和99%以上.适宜根状茎外植体愈伤组织诱导的培养基为MS+1.0 mg/L 6-BA+0.5 mg/L NAA,有利于增殖和丛生芽分化的培养基为MS+2.0 mg/L 6-BA+0.5 mg/L IBA和MS+3.0 mg/L 6-BA+0.1 mg/L NAA,而1/2MS+3.0～5.0 mg/L NAA适宜诱导试管苗生根培养.试管苗的移栽成活率可达85%以上.     

葶苈愈伤组织诱导及分化研究

为保护野生资源、实现人工栽培,本研究以葶苈(Draba nemorosa)嫩茎为材料,采用组织培养方法进行愈伤组织诱导与分化、不定芽生根与试管苗生根继代增殖培养,以及移栽和定植研究。结果表明,MS+6-BA 0.4 mg/L+2,4-D 2.5 mg/L是愈伤组织诱导培养和继代增殖培养的理想培养基;MS+6-BA 0.6 mg/L+NAA 0.1 mg/L 是愈伤组织分化培养和不定芽继代增殖培养的理想培养基;1/3MS+IAA 0.6 mg/L是不定芽生根培养和生根继代增殖培养的理想培养基;试管苗移栽成活率为86.8%,定植成活率为96.4%;定植苗保持了野生葶苈的植物学性状。     

‘SK4—316’胡萝卜体胚的诱导和培养

以'SK4-316'胡萝卜无菌苗的下胚轴为外植体,研究不同培养基配方和培养条件对愈伤组织诱导、体细胞胚间接发生及其同步化培养的影响,以及不同脱分化时间、脱分化培养基及外植体续存时间对体细胞胚直接发生的诱导及其培养的影响.结果表明:含3%蔗糖、0.8%琼脂的1/2MS + 2,4-D 2.5 mg/L + 6-BA(或KT)0.5 mg/L + CH 300 mg/L是诱导愈伤组织的良好培养基;1/2MS + 2,4-D 1.25 mg/L + KT 0.25 mg/L + 6-BA 0.25 mg/L(含3%蔗糖)适于愈伤组织分化并诱导体胚发生,0.02% ABA对体胚的诱导有促进作用,0.06% ABA或15% PEG能促进体胚成熟;外植体在MS + 2,4-D 1.0 mg/L固体培养基上脱分化培养48 h,再转入MS + CH 300 g/L液体培养基中可诱导体胚直接发生,但随着外植体续存于诱导培养基中时间的延长,体胚发生变异的几率也渐增.     

匙羹藤愈伤组织培养研究

以匙羹藤组培苗为外植体,研究茎段、茎尖和叶片以及不同植物生长调节剂组合对愈伤组织形成的影响;采用4因素4水平正交设计,用SPSS软件其结果进行分析。结果显示:各种外植体诱导愈伤的能力不同,依次是茎段、叶片。叶片诱导出非胚性愈伤组织,茎段、茎尖利于诱导胚性愈伤组织和胚状体;愈伤诱导的最佳培养基为MS+2,4-D 2.0 mg/L+6-BA 0.5 mg/L,诱导率为84.5%,愈伤生长旺盛,分裂快,颜色为淡黄色。愈伤组织分化成苗效果最好的条件是MS+NAA 1.0 mg/L+IBA 0.1 mg/L,分化率达到90.6%,最佳的生根条件是1/2 MS+NAA1.6 mg/L,生根率为100%。这为研究匙羹藤的组织的脱分化、分化以及胚状体的形成机制提供了参考。     

Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.)

Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system. Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material. In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium [Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8], followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days. The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb. After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds. After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting. We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%). Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants.     

罗汉果组培繁殖的技术要点

报道罗汉果组培繁殖的各项主要技术要点,包括组培条件、培养基的配制、外植体的选取与消毒、接种与培养、种源保存、炼苗与移栽、苗木包装与运输等。提出了5种培养基参考配方,即茎段诱导培养:MS+BA0.5～1.0mg/L+IAA(NAA)0.05～0.1mg/L+白糖3%+琼脂4.5g/L,pH5.8;茎尖诱导培养:MS+BA0.5～1.0mg/L+NAA0.05～0.1mg/L+椰子水100mL+白糖3%+琼脂4.5mg/L,pH5.8;继代培养(丛生芽方式):MS+BA0.3～0.7mg/L+NAA0.05/IAA0.1mg/L+白糖3%+琼脂4.5mg/L,pH5.8;继代培养(微型扦插方式):MS+BA0.1mg/L+IAA0.3mg/L+活性炭0.07g/L+白糖3%+琼脂4.5mg/L,pH5.8;生根培养:MS+BA0.07mg/L+IBA0.15mg/L+IAA0.1mg/L+活性炭0.1g/L+白糖3%+琼脂4.5mg/L,pH5.8。分析了外植体培养过程中可能出现的不良状况的原因并提出预防措施,明确了炼苗移栽的适宜条件并制定出相应的管理方法。形成了一套较为完整的罗汉果组培苗繁殖生产技术规程。     

芥蓝植株再生体系的优化

采用正交设计方法对影响芥蓝植株再生体系的因素进行了优化研究,结果表明：影响芥蓝植株再生的最主要因素是外植体类型,其次依次为NAA,BAP,蔗糖和AgNO3。结果进一步显示,最利于芥蓝再生植株的培养基条件为：MS BA P2mg／L NAA0．03mg／L 1％蔗糖 AgNO3 7．0mg／L 0．8％琼脂,最适宜的外植体类型为下胚轴,植株再生频率高达97．5％。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

Cellular origin and ultrastructural changes of regenerating shoots from tobacco (Nicotiana tabacum) internodes cultured in vitro

Ultrastructural changes were studied during shoot formation from tobacco internode slices cultured on Murashige and Skoog's (MS) basal medium plus 0.54 μ M naphthaleneacetic acid, 0.44 μ M 6-benzyladenine, 3% (w/v) sucrose and 0.8% (w/v) agar. Dramatic structural changes were observed in cortical cells below the internodal epidermis, especially those immediately centripetal to the stomata, by light and scanning electron microscopy. Transmission electron microscopic investigation revealed conspicuous structural changes to plastids at each stage during shoot regeneration. To confirm the significance of the cortical cells in shoot regeneration, protoplasts were isolated from them and shoots regenerated successfully. The ability of single cortical cell protoplasts to differentiate into shoots was demonstrated.     

应用正交设计建立青花菜植株的再生体系

通过L16(4 5)正交试验 ,研究最适合青花菜 2周龄下胚轴愈伤组织诱导和不定芽发生的植物生长调节物质的种类和浓度组合。结果发现在NAA、6 BA、TDZ和KT四种激素中 ,NAA对下胚轴愈伤组织发生指数、不定芽发生频率的影响作用最大 ,确定以MS +NAA 0 .2 5mg/L +6 BA 0 .2 5mg/L +TDZ 0 .0 1mg/L(琼脂 0 .8% ,蔗糖 3 % ,pH5 .8)作为单因子试验的培养基。NAA、6 BA和TDZ浓度的单因子试验结果表明最适合下胚轴的培养基配方为 :MS +NAA 0 .1mg/L +6 BA 1 .0mg/L +TDZ 0 .0 6mg/L(琼脂 0 .8% ,蔗糖 3 % ,pH5 .8)。     

Adventitious shoot regeneration of scotch spearmint (menthaxgracilis Sole)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Menthaxgracilis Sole (scoth spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 μM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, at TDZ 4.5 μM and 10% (v/v) coconut water and solidified with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% sucrose, and 0.054 μM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the first report describing adventitious shoot regeneration of scotch spearmint.     

Efficient plant regeneration from seedling explants of two commercially important temperate eucalypt species–Eucalyptus nitens and E. globulus

A reliable and reproducible method for plant regeneration in vitro of two important temperate eucalypts, Eucalyptus nitens and E. globulus, has been developed which utilises seedling explants. Highly regenerative callus was obtained from individual cotyledon and hypocotyledon explants of both species following cultivation on Murashige and Skoog’s (MS) basal nutrient medium supplemented with 30 g l⁻¹ sucrose, 5–10% (v/v) coconut water, 0.8% agar, 1 mg l⁻¹ -naphthalene-acetic acid (NAA) and 0.5 mg l⁻¹ N⁶ benzylaminopurine (BAP). Shoot differentiation was observed 7–8 weeks after transfer of callus onto regeneration medium containing 0.5 mg l⁻¹ NAA and 1 mg l⁻¹ BAP. In a few instances, direct shoot regeneration occurred without an intervening callus phase in both species. The frequency of plant regeneration was higher for callus derived from hypocotyl segments (30–35%) compared to cotyledonary explants (20–25%) though the average number of shoots per cotyledonary explant was generally higher than for hypocotyl explants. Somatic embryos were observed occasionally in E. nitens, arising from the surface of organogenic callus. Organised structures closely resembling somatic embryos were also observed in E. globulus. Regenerated shoots (30–40%) of both species could be rooted in modified MS media containing indole-3-butyric acid (IBA) and plantlets were successfully transferred to soil.     

In vitro culture of Cuminum cyminum regeneration of flowering shoots from calli of hypocotyl and leaf explants

Calli from hypocotyl explant of Cuminum cyminum L. (Cumin) grew rapidly on Gamborg's B₅ basal medium with the following supplements, (i) 0.5 mg/l — 2,4-D (ii) 4 mg/l — NAA plus 2 mg/l — Kinetin and (iii) 0.2 mg/l — NAA plus 0.2 mg/l — BAP, whereas calli from leaf explant in these media grew slowly. Hypocotyl and leaf calli produced roots when transferred to basal medium only and shoots in basal medium with 0.5 mg/l NAA and 0.1 mg/l BAP. Ninety percent of the shoots produced roots when they were transferred to half strength MS inorganic salts supplemented with 0.5 mg/l each of IBA and NAA.Fifty to sixty percent of rootless as well as rooted shoots produced terminal umbellate flowers on this medium.